Chemical signalling in the brain involves the regulated release of neurotransmitters into the synaptic cleft, where they bind to and activate specific cell surface receptors on the postsynaptic membrane. The long- term objective of the work proposed here is to understand how neurotransmitters modulate diverse cellular and physiological processes by interacting with multiple cell surface receptor subtypes. The experiments described in this application employ a combination of molecular genetic, biochemical, and electrophysiological methods to characterize drug binding and signaling transducing properties of specific neurotransmitter receptors in an effort to understand how they contribute to synaptic transmission in the vertebrate nervous system. These efforts are focused on two neurotransmitter systems that use serotonin (5-hydroxytryptamine, 5HT] and adenosine triphosphate [ATP] as endogenous agonists. Serotonergic neurons project to most regions of the central nervous system, where they regulate a wide variety of sensory, motor, and cortical, functions. These synapses are major targets for the action of psychotropic and antidepressant drugs like LSD, psilocybin, and Prozac and it is therefore assumed that transmission at these synapses plays an important role in the regulation of mood, behavior, and perceptual states. Drugs that modify transmission at serotonergic synapses are presently used in the management of depression, anxiety disorders, migraine headache, and chemotherapy- induced emesis. The role of ATP as an extracellular signaling molecule has more recently come under investigation, and its role in regulating a number of interesting and important physiological processes is now known to include fast excitatory neurotransmission in both central and peripheral synapses, mast cell degranulation, vasodilation, and stimulation of transepithelial ion transport in the lung. This latter effect may underlie the therapeutic effect of ATP in the treatment of cystic fibrosis-related lung disease. This research proposal is aimed at examining the function of excitatory ligand-gated ion channels that are activated by serotonin or ATP. The first aim is to use a cloned gene encoding a serotonin-gated ion channel, known as the 5HT3 receptor, to determine where agonists and antagonists bind to the 5HT3 receptor-ion channel complex. This project will involve the construction and functional analysis of specific receptor mutants, as well as the production of receptor protein in vitro for structure determination studies. The second aim is to use gene cloning and electrophysiological methods to identify novel receptor subunits that interact with a previously cloned 5HT3 receptor subunit, and to determine whether post-translational modification of the ion channel complex contributes to receptor regulation. The third specific aim is to use expression cloning methods to isolate and characterize a functional cDNA clone encoding the P2x receptor, an ATP-gated ion channel that is expressed in both neural and non-neural tissues.